The resistance of pathogenic bacteria to our most effective drugs is an ever-growing human health problem for which the only cure is the development of new antibiotics functioning with unique mechanisms of action. The transglycosylases, extracellular enzymes that synthesize the cell wall essential for the bacterial survival, represent an underexploited target in a pathway full of steps successfully inhibited by numerous antibacterials in clinical use. The identification and biochemical characterization of a new kind of transglycosylase will provide vital information that could be exploited in our search for novel therapeutics. Project Summary: Since the transglycosylases (TGs) are responsible for polymerization of the disaccharide building blocks of peptidoglycan, their activity is essential for bacterial survival and yet, deletion of all known TGs in B. subtilis and E. faecalis is not lethal. A unique kind of TG must exist in these and, most likely, many other organisms. Furthermore, the lack of appropriate biochemical tools for dissecting the mechanism of the "known" TGs has limited our ability to exploit these potential targets in the search for new antibacterial agents. The "missing" TG will be purified from extracts generated from a B. subtilis strain in which all known TGs have been deleted, using a TG activity assay to follow purification. Additionally, a unique photoaffinity cross-linking reagent based on the TG substrate, Lipid II, will be synthesized und utilized to help rapidly identify TG candidates. A list of TG candidate genes will be generated using LC/MS/MS sequencing, narrowed using a bioinformatics analysis, and confirmed by a combination of genetics and biochemical experiments. To understand the mechanism of this newly identified TG, an activity assay will be developed involving introduction of a single, unique radioactive probe to each glycan strand and separation of these strands by size-exclusion chromatography. This labeling method will allow us to, for the first time, easily and rapidly measure the average size of the glycan strands synthesized by TGs. In addition, we will be able to build a more detailed kinetic mechanism using this method to measure the rates of glycan polymer chain initiation and chain elongation. Finally, if labeled-Lipid II is a substrate for the TGs, then we can use this blocked substrate to determine the direction of elongation because it is unknown if these enzymes extend the glycan chain through addition of new units to the reducing or non-reducing end. The discovery of the first member of a new family of cell wall forming transglycosylases and the development of new tools to probe the TG mechanism will be important first steps towards understanding these underexploited targets for new antibacterial agents. [unreadable] [unreadable] [unreadable]